The hepatitis C virus (HCV) is an important cause of liver dysfunction which continues to spread in Thailand, particularly as genotype 6. The NS5B gene fragment is particularly variable and thus provides a valuable tracker for its spread. Therefore, the purpose of this study was to characterize the HCV genotype 6 based on partial NS5B region using Thai blood donor samples. Twenty-nine samples were genotyped as HCV 6 by nested PCR, nucleotide sequencing and amino acid sequence analysis. Amplified products were identified as HCV genotypes 6f, 6c, 6n, and 6i. There were amino acid variations of 4–18 residues in subtypes 6f, 6c, and 6n whereas subtype 6i was conserved when compared with their referent strains. In subtypes 6f, 6c, 6n, and 6i, the amino acid mutations at positions 244, 309, and 310 which are associated with HCV resistance were present. In summary, the sequences and phylogenetic analysis of NS5B of HCV used in our study yielded the genotypes 6f, 6c, 6n, and 6i. This finding indicates diversity of amino acids in NS5B of HCV. Characterizing the partial NS5B region among hepatitis C virus genotype 6 subtypes may predict efficacious anti-HCV therapy.